Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. Seasonal cholera outbreaks occur where the disease is endemic and can spread worldwide. V. cholerae[unreadable]s ability to cause epidemics is tied to its ability to survive in aquatic habitats. It has been proposed that V. cholerae[unreadable]s ability to form biofilms, matrix-enclosed, surface-associated communities, is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The objective of this proposal is to improve our understanding of biofilm matrix components, the mechanisms and regulation of biofilm formation, the mechanism of cyclic dimeric guanosine monophosphate (cdiGMP) signaling, and their importance in the biology of V. cholerae. In Aim 1, we will focus on characterization of biofilm matrix components. We will determine the structure of Vibrio polysaccharide (VPS) and the genes of the vps cluster required for VPS biosynthesis and biofilm formation. We will determine VPS binding capacities and localization patterns of the matrix proteins in biofilms, and test our hypothesis that these proteins bind to the sugars of VPS in order to stabilize the matrix. We will also investigate the enzymatic properties of a putative VPS lyase. Finally, we will ascertain the contribution of known biofilm determinants in V. cholerae pathogenesis. In Aim 2, we will investigate the regulation of biofilm formation. We will examine the regulatory features of genes required for biofilm matrix production and dissolution, and determine the interactions of the positive and negative transcriptional regulators with these promoters. We will also investigate the environmental conditions that influence the regulation of biofilm matrix production. In Aim 3, we will elucidate the molecular mechanisms by which cdiGMP signaling controls biofilm formation. We will determine the cellular localization of cdiGMP signaling proteins that modulate biofilm formation, and whether these proteins are compartmentalized in the cell. To identify the target proteins of the c-di-GMP signaling systems, we will search for proteins interacting with c-diGMP signaling proteins, as well as for c-diGMP receptor proteins within the rugose variants. We will then assess how these c-diGMP signaling proteins affect V. cholerae pathogenesis, using a murine infection model. Better understanding of the mechanism of biofilm formation, c-diGMP signaling, and the importance of both of these processes In V. cholerae biology will prove useful for the development of future strategies for predicting and controlling cholera epidemics, and to facilitate identification of novel drug targets for combating the pathogen during infection.